The genome assembly and annotation of the many-banded krait, Bungarus multicinctus

Snakes are a vital component of wildlife resources and are widely distributed across the globe. The many-banded krait Bungarus multicinctus is a highly venomous snake found across Southern Asia and central and southern China. Snakes are an ancient reptile group, and their genomes can provide important clues for understanding the evolutionary history of reptiles. Additionally, genomic resources play a crucial role in comprehending the evolution of all species. However, snake genomic resources are still scarce. Here, we present a highly contiguous genome of B. multicinctus with a size of 1.51 Gb. The genome contains a repeat content of 40.15%, with a total length exceeding 620 Mb. Additionally, we annotated a total of 24,869 functional genes. This research is of great significance for comprehending the evolution of B. multicinctus and provides genomic information on the genes involved in venom gland functions.


INTRODUCTION
Snakes are a fascinating group of reptiles that exhibit unique and diverse characteristics.
With approximately 3,070 extant species in all continents except Antarctica [1], they are known for lacking limbs, elongated body shapes, and an exclusively carnivorous diet.
Snakes have evolved many specialized adaptations, such as infrared sensing pits and a venom apparatus, which provide them with exceptional predatory capabilities [1]. These adaptations have made snakes important model organisms for evolutionary studies, yielding insights into limb development, sex chromosome evolution, and venom evolution.
In recent years, genetic approaches have become increasingly important in understanding the evolution and diversity of snakes [2]. By exploring the evolution of venomous snakes, we can gain a deeper understanding of the ecological and evolutionary roles of these intriguing species.
Bungarus multicinctus (NCBI:txid8616), also known as the many-banded krait or umbrella snake, is widely distributed throughout southern Asia, its range spanning across countries such as India, Pakistan, Indonesia, Sri Lanka, Malaysia, Bangladesh, Vietnam, and China [3]. B. multicinctus is recognized as one of the ten most venomous snakes in China, with a lethality rate ranging from 26.9% to 33.3% [4].
In this study, we collected a muscle sample of B. multicinctus to generate a highly contiguous genome with a genome size of 1.51 Gb. Its repeat element content reached 41.68%, providing new evidence for understanding the relationship between repeat elements and genome size in Elapidae species.

MAIN CONTENT Context
This study presents a highly continuous genome assembly of B. multicinctus. The genome size of B. multicinctus was found to be 1.51 Gb, with a GC content of 37.8% ( Table 1). The maximal scaffold length was 39.68 Mb, and the N50 length was 6.55 Mb, indicating a highly continuous genome sequence. This draft genome sequence of B. multicinctus will serve as an invaluable resource for further research on venomous snakes, enabling a better understanding of their genetic makeup.
The content of repetitive elements in our B. multicinctus genome was surprisingly large, reaching 41.68% with a total length of 675 Mb (Table 2). We analyzed the content of various repeating elements. While unknown types accounted for 51% of the repeating elements, LINEs and DNA transposons accounted for 10% and 8%, respectively ( Figure 1). Research indicates that although snake species have similar genome sizes, they exhibit significant differences in TE content, with low diversity in the types of TEs [5]. Specifically, species with a longer evolutionary history tend to have higher TE diversity [6]. Our results suggest that the significant expansion of repeating elements is an important manifestation of species differences.
We identified 24,869 functional genes of B. multicinctus and annotated them with Kyoto Encyclopedia of Genes and Genomes (KEGG). The majority of these genes were found to be involved in pathways related to Environmental Information Processing and Metabolism.
To construct a phylogenetic tree, we screened closely related species, including Anolis carolinensis, Chelonia mydas, Danio rerio, Deinagkistrodon acutus, Gallus gallus, Homo sapiens, Mus musculus, Ophiophagus hannah, Python bivittatus, Xenopus tropicalis, and Alligator mississippiensis. Our data is consistent with previous studies and can be used to construct a phylogenetic tree that clusters closely related species (Figure 4) [8].   (RRID:SCR_017979) [11]. In addition, genomic DNA was isolated using the AxyPrep genomic DNA kit (AxyPrep, USA) for whole-genome sequencing.
We extracted the total RNA using the TRlzol reagent (Invitrogen, USA) following the manufacturer's protocol. RNA quality, purity, and quantity were assessed using the
The genome completeness was evaluated by analyzing sets of BUSCO v5.2.2 using genome mode and lineage data from vertebrata_odb10 [27], following the standard scientific methodology.

REUSE POTENTIAL
Venomous animals have fascinated and influenced humans since ancient times, and the venom gland is a special evolutionary mechanism that snakes have developed to adapt to their ecological environment [30]. In recent years, ecosystems have changed due to climate variations, and toxic species threaten not only humans but also native species and livestock [31,32]. Therefore, it is crucial to collect genomic resources of venomous snakes and explore the formation mechanism of venom glands and venom production.
Genome assemblies of reptiles, including snakes, have always been challenging to generate. However, Xu et al. recently published an article on the origin of neurotoxins in the Elapidae family based on a high-quality genome assembly of the many-banded krait [29]. Using third-generation sequencing and Hi-C, Xu et al. assembled the many-banded krait genome to the chromosome level, achieving a BUSCO score of 94.6% and a scaffold N50 of 149.80 Mbp. Our assembly resulted in a BUSCO score of only 90.9%.
Although our assembly did not achieve the same level of genome continuity as Xu et al., we obtained a relatively complete genome of the many-banded krait using stLRF second-generation sequencing data. Being sampled from a different geographic location provides a genomic resource for future research exploring the evolution and origin of reptilian species, including snakes.
Our data can be combined with already published and new venomous snake genome data to reconstruct the evolutionary history of venomous snakes and other reptiles. Our genome data can also be used in venomics research to explore toxic gland genes and the mechanism of toxic gland production.

DATA AVAILABILITY
The data that support the findings of this study have been deposited into CNGB Sequence Archive (CNSA) [33] of China National GeneBank DataBase (CNGBdb) [34] with accession number CNP0004003. The data are also hosted in NCBI with accession number PRJNA934116. Additional data is available in the GigaDB repository [35].

Consent for publication
All experimental procedures were approved by the Institutional Animal Care and Use Committee of Northeast Forestry University (2023048).